The present invention is directed to a fluorescent imaging system.
There are currently numerous methods for fluorescent imaging, all of which have as their objective the illumination of a sample with excitation light of one wavelength while imaging a resulting fluorescent emission at a second, longer wavelength. Because the fluorescent efficiency of many samples is low, i.e. typically 1 photon of fluorescent emission or less per 100 photons of excitation, the optical imaging system must efficiently collect the weak fluorescent emission without interference from the much stronger excitation signal. The optical system must provide an efficient optical path for delivering emission light to the image, but little or no such path for excitation light. Typically, spectral filters, such as colored-glass or interference filters, are used to provide at least some degree of the required wavelength selectivity which is enhanced through a careful choice of the overall optical design.
Prior art optical systems normally incorporate one optical path for the excitation light and a second optical path for the fluorescent emission. These optical paths necessarily overlap at the sample, and in many systems the two paths also make common use of one or more of the optical elements.
Two approaches are widely used to minimize the coupling of light from the excitation to the emission optical path. The first approach is to illuminate the sample with light having a range of angles to which the emission optics are non-responsive. One method for achieving this is illuminating the sample from the side while collecting the fluorescent emission from the top. A portion of the fluorescent emission, which is more or less isotropic in angular distribution, is captured by the collection optics while the angularly-restricted excitation beam leaving the sample proceeds, uncollected, to a baffled optical trap.
Although such an arrangement can be used successfully for single-point measurements, it is not generally suitable in imaging applications. Imaging systems commonly use "dark-field" illumination, in which diaphragms, or zone plates, in the illumination and collection objectives insure that the sample is illuminated over a first selected cone of angles while the emission is collected over a second, but different, cone of angles. An inherent feature of the dark-field method is that the effective numerical aperture ("NA") of the objectives is reduced, causing a significant and undesired reduction in optical efficiency. Moreover, by reducing the NA the diffraction limit of the instrument is degraded so that both image quality and resolution also deteriorate.
The second known approach to minimizing coupling of light from the excitation to the emission path is separating those two optical paths through the use of a dichroic beamsplitter. Most widely employed is the "epi-illumination" method, which utilizes a dichroic beamsplitter that strongly reflects light at the excitation wavelength, but transmits light at the emission wavelength. The beamsplitter is oriented to reflect light from the illumination optics into a common objective through which it illuminates the sample. Fluorescent emission, collected by the same objective, passes through the dichroic beamsplitter without significant loss and proceeds along the remainder of the emission optical path. Since the beamsplitter provides low transmission at the excitation wavelength, little of the stray excitation light that is reflected or scattered by the sample finds its way into the emission optical path. Unlike the dark-field arrangement, there are no limitations on the NA of the objective, and it is possible to use objectives with a high NA to achieve high throughput and high image resolution.
However, in the epi-illumination method the dichroic beamsplitter inherently restricts one to a single set of excitation and emission wavelengths since the beamsplitter affords high reflection at a particular predetermined band of excitation wavelengths and high transmission at another particular predetermined band of emission wavelengths.
While it is possible to design a dichroic beamsplitter which provides for three or even four excitation bands and a corresponding number of emission bands, for several reasons such beamsplitters offer only a limited increase in versatility. First, the optical performance of a multi-band device is generally inferior to that of a single-band device due to limitations in the optical coating art. Second, the wavelengths of the various bands cannot be independently specified or selected due to constraints in the thin-film coating art. However, each particular fluorescent species has a spectral response which dictates the use of an optimal band for excitation and, accordingly, an optimal emission band. In practice, the various bands reflected and passed by the beamsplitter cannot all be chosen for maximum efficiency of excitation and collection, with the result that for one or more fluorescent species, the system is inefficient at the excitation fluorescence and/or at the fluorescent emission signal wavelengths.
A third drawback of multiband beamsplitters is that any given wavelength must be dedicated to either excitation or to emission. If dedicated to excitation, the dichroic beamsplitter must be highly reflective, whereas for emissions it must be highly transmissive. Thus, it is fundamentally impossible with a dichroic beamsplitter to observe fluorescent emission at any wavelength which is or may be used as an excitation band. This presents a severe restriction in attempts to devise a system for imaging multiple fluorescent species. It is undesirable to mechanically exchange the beamsplitter to overcome this restriction because this leads to vibration and image shift in the system.
In addition, the restrictions imposed by a dichroic beamsplitter severely restrict spectroscopic imaging systems. When more than one fluorescent species is present, the emission spectra may overlap and the observations at a single wavelength may not uniquely identify the emitting species. By taking a complete spectrum and resolving it into relative contributions from the different species, each of which has a characteristic spectral shape, the presence and quantity of each species can be accurately determined. However, this requires a fluorescence imaging system to obtain a spectrum at many wavelengths. Although it is ideally desirable to accommodate a continuous unbroken spectrum over the full range of emission, such an objective is not achievable by the use of a dichroic beamsplitter which merely dedicates specific fixed wavelength bands to excitation and others to emission.
Thus, there is currently no fluorescent imaging system that accommodates the use of high and unrestricted NA in the objectives and which does not impose severe limits on the spectral location of the excitation and emission bands that are employed for imaging multiple fluorescent species, or from spectroscopic imaging of fluorescent species.